Complete chloroplast genome of Angelica hirsutiflora Liu et al. 1961 (Apiaceae)

Abstract Angelica hirsutiflora Liu et al.1961, is a perennial herb in the Apiaceae family that is endemic to Taiwan. In this study, the complete circular chloroplast genome of A. hirsutiflora was reconstructed and annotated using Illumina sequencing. The size of the chloroplast genome is 154,266 bp, consisting of two inverted repeats (IRs, 25,075 bp) separated by a large single-copy region (LSC, 86,569 bp) and a small single-copy region (SSC, 17,547 bp). The GC content of the chloroplast genome is 37.6%. There are 114 different genes in the chloroplast genome of A. hirsutiflora, including 80 protein-coding genes, 30 tRNA genes and four rRNA genes. A maximum-likelihood phylogenetic analysis showed that A. hirsutiflora forms a distinct clade, and separated from other species within the genus Angelica. This study provided insights into the evolutionary relationships among different species of Angelica.


Introduction
Angelica hirsutiflora Liu et al. 1961, is a perennial herb in the Apiaceae family that is endemic to Taiwan (Liu et al. 1961;Liu and Kao 1977;Kao 1993).It is mainly distributed in the coastal areas of northern Taiwan (Figure 1).Angelica hirsutiflora is considered an endangered species due to overexploitation and habitat destruction by humans (Editorial Committee of the Red List of Taiwan Plants 2017).Angelica hirsutiflora was formally descripted in Liu et al. (1961).Yamazaki (1990) treated A. hirsutiflora as a variety of Angelica japonica A. Gray, 1859, namely, A. japonica var.hirsutiflora (Liu, Chao & Chuang) T. Yamaz. 1990, which is distributed in the Ryukyu Islands and Taiwan.Seo et al. (2005) concluded that A. hirsutiflora can be taxonomically treated as independent species using allozyme analyses.According to Liao et al. (2022), A. hirsutiflora belongs to the littoral Angelica clade and is closely related to A. japonica.Therefore, we adhered the treatments of Kao (1993), Liu et al. (1961) and Liu and Kao (1977), which consider A. hirsutiflora a distinct taxonomic species.Recently, plastid phylogenomics has provided a tool to improve the reconstruction of phylogenetic relationships in Angelica L. (Wang et al. 2021).The chloroplast genome of A. hirsutiflora, obtained using next-generation sequencing, is characterized and assembled for the first time.This study enhances our understanding of the phylogenetic relationships among different species of Angelica.

Plant collection
A wild individual of A. hirsutiflora was collected from Jinshan District (121 � 37'58"E, 25 � 15'36"N), New Taipei City, Taiwan.Despite being regarded as an endangered species, it is not a legally protected species in Taiwan.The collection location in this study is not a privately owned or protected area.No permits were required for this study.A specimen was deposited at the herbarium of the Endemic Species Research Institute (TAIE) (https://www.tesri.gov.tw/,Tsai-Wen Hsu, twhsu@tesri.gov.tw) under voucher number No. 24169.

DNA extraction, sequencing, assembly, and annotation
Total genomic DNA was extracted from the leaf materials of A. hirsutiflora using CTAB extraction.The library was sequenced by the Illumina NovaSeq 6000 platform with the double terminal sequencing method (pair-end, 150 bp).Paired-end reads were assembled using GetOrganelle v. 1.7.7 (Jin et al. 2020).Annotation of the chloroplast genome was performed using GeSeq (Tillich et al. 2017).The gene graphical map of the chloroplast genome was constructed using CPGView (http://www.1kmpg.cn/cpgview)(Liu et al. 2023).The annotated genomic sequence has been deposited in GenBank under accession number OQ773546.

Chloroplast genome features of A. hirsutiflora
A total of 14.7 Gb of clean data were generated.The structure of the chloroplast genome of A. hirsutiflora was circular, and the size was 154,266 bp, with an average depth of 441.79 x (Supplementary Figure 1).It was composed of a pair of inverted repeat regions (IRs: 25,075 bp) separated by a large single-copy (LSC) region of 86,569 bp and a small single-copy (SSC) region of 17,547 bp.The GC content of the chloroplast genome was 37.6%.There were 114 different genes in the chloroplast genome of A. hirsutiflora, including 80 protein-coding genes, 30 tRNA genes and four rRNA genes (Figure 2).Furthermore, 16 (ten protein-coding and six tRNA genes) genes contained one intron, and three proteincoding genes (clpP1, pafI and rps12) contained two introns.rps12 has been recognized as a trans-splicing gene (Supplementary Figure 2).

Phylogenetic analysis
The phylogenetic results showed that Angelica species formed a monophyletic clade, which was mainly divided into two groups (Figure 3).Angelica hirsutiflora formed an independent clade to other species of the genus Angelica.This study provides the chloroplast genome information of A. hirsutiflora, which can be used for species identification and phylogenetic analysis within Angelica species.

Discussion and conclusion
Compared to other species of the littoral Angelica clade in Taiwan, A. hirsutiflora is the only species that is distributed in coastal areas, implying a possible distant relationship.The chloroplast genome of A. hirsutiflora was assembled and annotated for the first, and the genome was determined to be 154,266 bp in length, containing 114 genes.Angelica hirsutiflora formed a distinct clade, and separated from other species within the genus Angelica in the phylogenetic tree.Liao et al. (2022) and Wang et al. (2024) proposed that A. hirsutiflora is closely related to A. japonica according to nrITS sequences.Angelica hirsutiflora morphologically resembles A. japonica (Seo et al. 2005).However, the information of chloroplast genome of A. japonica is absent in the NCBI database.To better understand its phylogenetic relationships, additional chloroplast genomes from the littoral Angelica clade are urgently needed.This study has contributed to the enlargement of the chloroplast genome database for Angelica and has provided valuable insights into the evolutionary relationships among various Angelica species.

Figure 2 .
Figure 2. Schematic map of overall features of the chloroplast genome of A. hirsutiflora.Genes outside the circle are transcribed clockwise, while genes inside the circle are transcribed counterclockwise.Different functional groups are represented by different colors.The darker and lighter gray in the inner indicated the GC and at content, respectively.